The present invention is in the field of the delivery of therapeutic agents, and more particularly in the area of enhancement of transport or delivery of molecules into the cell cytosol, through cell barriers or layers of cells, or through lipid membranes, using membrane barrier transport enhancing agents alone or in combination with a stimulus and/or enhancer that modifies the structure and/or properties of the agents.
This application claims priority to U.S. Ser. No. 60/070,411 entitled xe2x80x9cmembrane Disruptive Agentsxe2x80x9d filed Jan. 5, 1998, by Allan S. Hoffman, Patrick Stayton, and Oliver Press.
Specific, efficient delivery of therapeutic and diagnostic compounds to cells is a major goal of most pharmaceutical companies. A number of different approaches have been utilized to increase specificity and uptake. The most common has been to target the therapeutic or diagnostic agent to specific types of cells by conjugation of the agents to antibodies that recognize antigens specifically or predominantly associated with the cells. Other agents, such as polycationic complexes, liposomes, and lipid complexes, have been employed to increase uptake of compounds generally by cells.
There are several therapeutic agents which are only effective if they are delivered intracellularly, including genetic material and various proteins. Gene therapy requires the intracellular delivery of genetic material to treat genetic disorders, cause mutations in the genetic material in various cells, such as tumor cells, and bind to or interact with various sites in the cells to cause an effect. Examples of proteins include toxins which are only poisonous once they have been released from the endosome into the cytoplasm. To increase their specificity, immunotoxins have been prepared that include the toxin conjugated to an antibody that targets tumor-associated antigens. Immunotoxins have had limited success as therapeutics, however, in part due to the inadequacy of penetration into tumor nodules and ineffective delivery of the toxin into cytosolic ribosomes.
It is often difficult to deliver compounds, such as proteins, genetic material, and other drugs and diagnostic compounds, intracellularly because cell membranes resist the passage of these compounds. Various methods have been developed to administer agents intracellularly. For example, genetic material has been administered into cells in vivo, in vitro and ex vivo using viral vectors, DNA/lipid complexes and liposomes. DNA has also been delivered by synthetic cationic polymers and copolymers and natural cationic carriers such as chitosan. Sometimes the synthetic polymers are hydrophobically modified to enhance endocytosis. While viral vectors are efficient, questions remain regarding the safety of a live vector and the development of an immune response following repeated administration. Lipid complexes and liposomes appear less effective at transfecting DNA into the nucleus of the cell and may potentially be destroyed by macrophages in vivo.
Receptor mediated endocytosis offers an alternative means to target specific cell types and to deliver therapeutic agents intracellularly. Receptor-mediated endocytosis (RME) occurs when ligands bind to cell surface receptors on eukaryotic cell membranes, initiating or accompanying a cascade of nonequilibrium phenomena culminating in the cellular invagination of membrane complexes within clathrin-coated vesicles. Compounds which interact with specific cell surface receptors are employed to target specific cell surface receptors. The compounds are endocytosed into the endosomes once the compounds interact with the cell surface receptors. Linkages have been made directly with the compounds, or, in the case of DNA, through conjugation with polycationic polymers such as polylysine and DEAE-dextran which are then complexed with the DNA. Haensler et al., Bioconj. Chem., 4:372-379 (1993).
Even after therapeutic agents are delivered intracellularly, normal trafficking in the cell can minimize their effectiveness. For example, certain antibody-antigen conjugates are readily endocytosed. However, after endocytosis, the antibody is not released into the cytosol but rather remains isolated in endosomes until it is trafficked to a lysosome for degradation. Press, O. W. et al., Cancer Research, 48: 2249-2257 (1988). Endosomes are membrane bound phospholipid vesicles which function in intracellular trafficking and degradation of internalized proteins. The internal pH of the endosomes is between 5.0 and 5.5. A toxin conjugated with this antibody will be similarly isolated in the endosome, and, if trafficked to a lysosome, will be rendered ineffective. Genetic material, being negatively charged, is often complexed with polycationic materials, such as chitosan and polylysine, for delivery to a cell. Both immunotherapy and gene therapy using polycation/nucleic acid complexes are limited by trafficking of the complexes by the cell from endosomes to lysosomes, where the antibody conjugates or nucleic acids are degraded and rendered ineffective.
Accordingly, a major limitation of many potentially useful therapies is that the agents, even if they can be targeted to the desired cells and endocytosed by the cells, often are not effectively released from endosomes into the cytosol, but are degraded by lysosomes.
Several methods have been proposed to avoid or minimize lysosomal degradation of these agents. One method involves including lysosomotrophic agents such as chloroquine in formulations used to administer therapeutic agents intracellularly. Another method involves disrupting the endosome so that the agent is delivered into the cytosol before it is transported to and degraded by the lysosomes. It is preferable to disrupt the endosome so that the material never comes in contact with the lysosomes. At least two pathways have been developed for disrupting the endosomal membrane. One method takes advantage of the pH inside the endosomes, and uses materials which are relatively hydrophilic at physiological pH (around 7.4) and relatively hydrophobic at the pH inside of the endosomes. Examples of such materials are carboxylic acid containing polymers such as the hydrophobic polyacid poly(2-ethylacrylic acid) (PEAA), which are negatively charged at alkaline pH and uncharged at the pH inside the endosome due to protonation of the carboxylic acid moieties.
PEAA has been shown to solubilize lipid membranes in a pH dependent manner, permeabilizing and solubilizing membranes at an acidic pH (approximately 6.3), while having no effect at alkaline pH. Thomas, J. L. et al., Biophysical Journal 67:1101-1106 (1994); Thomas, J. L. et al., Acc. Chem. Res., 25: 336-342 (1992). It has been postulated that the effects of PEAA are due to its amphiphilicity rather than structure, consistent with a hydrophobically driven micellization process. A similar process has been hypothesized for the interaction of apolipoproteins, melittin, and other amphiphilic xcex1-helix based polypeptides with lipid membranes.
Various peptides also disrupt endosomal membranes in a pH dependent manner. Examples of peptides shown to disrupt liposomes, erythrocytes, and endosomes, include viral peptides, such as influenza virus peptides and peptides that include the 23 amino terminal amino acid sequence of influenza virus hemagglutinin, and related peptides which viruses destabilize endosomal membranes in a pH dependent manner such as GALA (also known as EALA) which includes repeating glutamic acid-alanine-leucine-alanine blocks. These peptides have been conjugated with DNA complexes that utilized a receptor mediated endocytosis pathway for uptake into cultured cells. A strong correlation was observed between pH specific erythrocyte disruption and gene transfer. Plank, C. et al., J. Biol. Chem. 17(269):12918-12924 (1994); Hughes, J. A. et al., Pharm Res., 13(3):404-(1996). Other peptides include melittin and derivatives, which are membrane channel formers. Pawlak, M. et al., Protein Science 3:1788-1805 (1994). GALA has been conjugated with a polycationic polymer (polyamidoamine cascade polymers, dendritic polymers synthesized from methyl acrylate and ethylenediamine), and the polycationic polymeric block has been complexed with plasmids encoding reporter genes. Haensler, J. et al., Bioconj. Chem., 4:372-379 (1993).
None of these methods or materials have solved the transport or delivery problems. It would therefore be advantageous to provide improved compositions for delivering diagnostic and/or therapeutic agents to the cytoplasm of a cell without significant lysosomal degradation.
It is another object of the present invention to provide compositions for enhanced transport of diagnostic or therapeutic agents, including proteins and genetic material, or other molecules through other cell membranes, cell barriers or cell layers, or through lipid membranes.
It is a further object of the present invention to provide such compositions that can be controlled and manipulated externally, for example, using non-invasive means such as ultrasound to enhance delivery or transport.
Compositions and methods for transport or release of therapeutic and diagnostic agents or metabolites or other analytes from cells, compartments within cells, through cell layers or cell barriers, or lipid membranes are described. The compositions include a membrane disruptive agent or xe2x80x9cmembrane barrier transport enhancing agentxe2x80x9d and are usually administered in combination with an enhancer and/or exposure to stimuli to effect disruption, transport or release. In a preferred embodiment, the compositions include compounds which disrupt endosomal membranes in response to the low pH in the endosomes but which are relatively inactive toward cell membranes, coupled directly or indirectly to a therapeutic or diagnostic agent. Other disruptive stimuli can be used with the membrane barrier transport enhancing agent, such as light, electrical stimuli, electromagnetic stimuli, ultrasound, temperature, or combinations thereof. The compounds can be coupled by ionic, covalent, hydrophobic or H bonds to an agent to be delivered, to a ligand which forms a complex with the agent to be delivered, or to a carrier. Agents to be delivered can be therapeutic and/or diagnostic agents, including proteins or peptides, synthetic organic molecules, nucleotides or oligonucleotides, carbohydrates, metals, radiolabels, or combinations thereof.
In a preferred embodiment, the endosomal membrane disrupting compounds are polymers, most preferably pH sensitive polymers which are inert at physiological pH (around 7.4) but which disrupt the endosomal membrane at the pH range inside the endosome (between about 5.1 and 5.5). Suitable polymers include poly(alkyl)acrylic acids, cationic polymers, copolymers of the polymers with pH sensitive proteins and/or peptides which can disrupt endosomes at the pH range inside the endosomes, and copolymers with peptides which contain imidazole groups and/or other groups which are known to disrupt endosomal membranes. Optionally, the compositions can include compounds which minimize lysosomal function, enhance endocytosis or target the compositions to particular cell types. Alternatively, or in addition, the composition can include ligands such as polycationic materials like polylysine or chitosan, which form a complex with the agent to be delivered, stabilizing the agent and in some cases further enhancing endocytosis by causing membrane disruption. The compositions can also include a carrier, for example, nanoparticles or microparticles, liposomes or lipid vesicles. The lipid vehicles, especially cationic liposomes, may themselves cause membrane disruption. The membrane disrupting agents can be incorporated onto, into or within these carriers. The compositions can be administered systemically or locally using known methodologies, in an amount effective to diagnose or treat a patient in need thereof. The materials are particularly useful for delivery of genetic material to cells in vitro, for example, for gene therapy. The compositions are also useful for manipulation of other types of cells such as bacterial cells, which can be readily exposed to an external stimuli to cause membrane disruption, including changes in pH.
Treatments which enhance delivery can also be used with the membrane disrupting agents. In a particularly preferred embodiment, ultrasound is used to enhance delivery or transport into or out of cells or through the skin. This is useful not only for drug delivery, but also transport of analytes such as glucose, which can then be measured and the amount present in the interstitial fluid correlated with blood levels. This treatment is also particularly useful for gene therapy into other cell types such as endothelial and smooth muscle cells, especially in the arterial environment, for example, for the treatment or prevention of restenosis. The treatment can be applied to the site before, at the time of, or following administration of the membrane disrupting agent. One advantage of ultrasound is that the membrane disrupting agent, preferrably targeted to specific cells, can be administered systemically, allowing time for the agent to travel to a distal location, followed by administration of the ultrasound. The preferred type of ultrasound is high intensity focused ultrasound (HIFU). The ultrasound can be delivered by a variety of means, including the direct application of the transducer to the surface of the tissue to be treated, or at some distance removed from the tissue surface, in which both plane waves and focused acoustic waves can be utilized. Optimal frequences typically range from a 20 kHz to 10 MHz, preferably less than 3 MHz.